GPCR Signaling in the Genesis and Progression of Pancreatic Cancer

Abstract

Ductal adenocarcinomas of the pancreas are the 4th most common cause of cancer death. The 1 and 5 year survival rates for all stages combined are currently 26% and 5% respectively. Median survival is less than 6 months. Despite remarkable progress in the fields of genetics, cancer biology, and advances in surgical techniques as well as chemotherapeutics, our ability to recognize and treat patients with pancreatic cancer remains poor. GPCR signaling modules have been increasingly implicated in the genesis and progression of pancreatic cancers. Aberrant agonist production, receptor expression and dysfunctional signaling resulting from genomic instability in a background of a heterotopic tumor-stromal microenvironment, contribute to the initiation, progression, and eventual metastasis of the disease. Numerous GPCR agonists, including lysophosphatidic acid (LPA), along with their cognate receptors have been implicated in this oncogenic process. LPA, one of the simplest bioactive lipids, has been shown to be a potent stimulant of metastatic behavior in in vitro models. It also acts as a mitogen by inducing proliferation and cell survival pathways in various normal and transformed cell lines. In patients with pancreatic cancer both the receptors and ligand have been found to be overexpressed. It has been noted that pancreatic cancer cell lines expressing higher levels of the LPA receptors present with greater motility. This has led to the hypothesis that LPA contributes to the progression of pancreatic cancer through the promotion of a metastatic phenotype. However, the underlying mechanisms have not been well described. LPA receptors have been shown to couple to the Gi, Gq, or G12 family of heterotrimeric G proteins. Consequently, signals transduced through these receptors have been shown to stimulate Gαi, Gαq, and Gα12/13 dependent pathways. While earlier studies have linked Gαi to LPA induced migration, there is recent evidence to suggest that Gα13 may provide a major signaling mechanism for LPA receptors stimulating migration in diverse cell types including cancer cell lines. Given the ominous nature of pancreatic cancers it is of critical importance to understand the mechanisms that promote more malignant phenotypes and to assess the role of Gα13 in this process. The goal of this thesis therefore is to define the role of Gα13 in LPA-mediated migration of pancreatic cancer cells. To assess the oncogenic potential of LPA and the role of Gα13 in stimulating the migration of pancreatic cancer cells, a panel of pancreatic cancer cell lines was assembled and characterized with regard to their expression of the LPA receptors as well as the Gα subunits of the heterotrimeric G proteins. These cell lines were further studied through a series of proliferation, wound healing, and transwell migration assays to assess the role of LPA in the induction of proliferation and migration in pancreatic cancer cells. The results demonstrated that LPA functions as a mitogen in certain pancreatic cancer cell lines, but is a potent stimulant of cell motility and invasive migration. Interestingly, these studies indicated that this response proceeds through routes that may not involve Gαi, as a potent migratory response was observed in MDAPanc28 cells which lack expression of the Gαi subunit. This was verified through the transwell assays conducted in the presence of PTX demonstrating that migration occurs independently of PTX sensitive mechanism and thus independently of Gαi.. Using a dominant negative mutant strategy, the studies presented in this thesis establishes the role of Gα13 in mediating LPA-LPAR stimulated migration of pancreatic cancer cells. Using pancreatic cancer cell lines that stably express the competitively inhibitory dominant negative mutant of Gα13, the ability of these mutants to inhibit a LPA mediated migratory response was monitored by wound-healing as well as transwell migration assays The results of these studies indicated a substantial attenuation of the migratory response and demonstrated for the first time the critical role of Gα13in LPA induced migration in a pancreatic cancer cell line.